Fluorescent probes used to detect important biological events in living cells or animals have been in increasing demand in the biological and biomedical fields over the past two decades. Many kinds of fluorescent bioprobes have been developed, such as organic dyes, inorganic nanoparticles, and fluorescent polymers.
Traditional commercially available organic dyes, such as fluorescein isothiocyanate (FITC), rhodamine, propidium iodide, ethidium bromide, and nile red, are highly emissive in dilute solutions but become weakly fluorescent or even non-emissive in the aggregated state. This problem, termed aggregation-caused quenching (ACQ), has seriously obstructed the advancement of fluorescent sensors. It has been attributed to the nonradiative decay of sandwich-shaped excimers and exciplexes formed among the closely packed dye molecules in the aggregates. A low dye concentration staining the cell may be free of aggregation but can only offer weak emission, and the small number of dye molecules that enter into the cell may be easily photobleached during the imaging process. Therefore, the fluorescence emission can be further weakened, rather than enhanced, if more fluorophores are loaded into the cell due to the ACQ effect. Accordingly, the dyes are usually used in trace amounts (often at nM level).
Such problems can be avoided by the use of inorganic quantum dots (QDs), which are highly fluorescent and resistant to photobleaching. However, QDs need improved hydrophilicity and reduced toxicity, as they are usually composed of heavy metals and chalcogens (e.g. CdS, CdSe, ZnSe, and PdTe), which are well known toxicants or carcinogens.
Another approach to mitigate the ACQ effect of traditional organic dyes is labeling them onto macromolecular chains to form fluorescent polymers. Although macromolecular chains could alleviate the aggregation of fluorophores due to the obstructing effect of macromolecular segments, they are still inclined to aggregate at a high concentration due to their hydrophobic aromatic cores. Accordingly, the ACQ effect and toxicity of fluorescent probes are constant problems in the development of fluorescent bioprobes to detect important biological events in living cells or animals.
Likewise, fluorescent labeling of biomolecules such as proteins and DNA has been attractive for both biomolecular tracing in biological processes and detection and quantization of biomolecules. For biomolecular tracing, the labeling products should be stable for long-term tracing. In addition, the attraction of fluorescent dyes to biomolecules has to be eliminated through a mild labeling protocol with desirable degree of labeling (DOL) so that the labeled biomolecules can maintain their natural folding structures and biological activities. On the other hand, sensitivity of the biomolecule analysis on gel usually depends on the solubility of the stained biomolecules or the background intensity of the stained gel.
Two main methods have been developed for covalent modification of proteins through chemical reactions between reactive groups of fluorescent dyes and special amino acids of proteins. Amine-reactive fluorescent dyes such as fluorescein isothiocyanate (FITC), Cy3 and Cy5 were applied to label lysine (Lys) residues of protein samples and used successfully in immunostaining assays or protein detection on gel.
Thiol-containing proteins were found to play important roles in the biochemical functioning of cells. The amino acid cysteine (Cys) is concerned in catalytic or oxidation/reduction functions of peptides and proteins of which it is a component. A commonly used method is labeling of cysteine (Cys) residues of proteins with alkylating agents.
Fluorescent DNA segments have attracted great interest because of their applications as fluorescent probes for gene detection through fluorescent in situ hybridization (FISH) or hybridization in solutions with the target nucleic acids. Fluorescent dyes can be coupled with an amine or thiol modified nucleoside, nucleotide or oligonucleotide. The fluorescent products can be used to synthesize fluorescent DNA strands for detection of genetic materials in vivo and in vitro.
Fluorescent labeling of biomolecules with water soluble dyes through their charge-charge interaction also has many advantages such as easy handling and fast detection.
However, all the binding aggregation-caused-quenching (ACQ) dyes face the self-quenching problem when the fluorophore to protein ratio (F/P ratio) is over a certain level. Moreover, the poststaining method is employed by most traditional fluorescent dyes while protein labeling requires the labeled proteins to be fixed with dilute acetic acid. This inhibits the transfer of the proteins on the gel to the nitrocellulose membranes for further analysis during western blotting. Also, the DOL of the fluorescent DNA prepared with traditional fluorescent dyes must be controlled at a relatively low level to avoid quenching of fluorescence, and thus the sensitivity of the probes is significantly weakened.
Accordingly, there is a great need for the development of fluorescent bioprobes that exhibit aggregation induced emission (AIE). In addition, there is a need for fluorescent bioprobes that are not limited by the F/P ratio restriction, thereby allowing the use of relatively high concentrations of fluorophores in both prestaining and poststaining methods. Furthermore, there is a need for fluorescent bioprobes that are highly biologically compatibility, nontoxic to live cells, do not interfere with the cell physiology and proliferation, resistant to photobleaching, and have the ability to stay inside live cells for a long period of time without leaking out into the culture media.